N-terminal Parathyroid Hormone Research Articles

MON-537 Fusion of Parathyroid Hormone to Growth Hormone Binding Protein Delays Clearance Whilst Retaining Biological Activity

Parathyroid hormone (PTH) is an 84 aa peptide and biological activity resides in residues 1-34. PTH 1-84 (Natpara) is licensed for treatment of hypoparathyroidism replacement but requires daily injections and is complicated by fluctuating calcium levels. There is an unmet need for a long acting PTH that provides constant physiological levels of PTH bioactivity. We have tested whether fusions of human PTH 1-34 to growth hormone binding protein (GHBP) could generate long acting PTH molecules. Two PTH fusion molecules were constructed: Fusion-1 consists of PTH (1-34) linked to GHBP (residues 1-238), and Fusion-2 consists of human PTH (1-34) linked to the N-terminal PTH receptor domain (PTHrExt, residues 29-187) and GHBP (residues 1-238). Fusions were expressed as secreted products from a CHO cell line and purified using a combination of ion exchange and affinity chromatography. In vitro bioactivity was measured using a Dual Luciferase Reporter Assay (DLRA), using the rat osteoblast-like cell line, UMR-106. Both fusions showed biological activity when compared to PTH 1-34 with an EC50 nM (mean ± SD) for PTH 1-34 of 40 ± 15.66, Fusion-1, 151 ± 5.85, & Fusion-2, 721 ± 307. PTH was 3.8-fold more active than Fusion-1 and 18-fold more active than Fusion-2; although, Fusion-2 showed a consistently higher maximal fold induction compared to both PTH and Fusion-1. A pharmacokinetic (PK) study for Fusion-1 was conducted in Crl:WI(Han) male rats (n = 6) by administering a single subcutaneous bolus injection at 10 nmol/kg. Serum samples were analysed for the presence of Fusion-1 using an in-house Elisa. A Cmax (mean ± SD) of 1.16 ± 0.36 nM was observed at 8 hrs with a serum half-life of 36 hrs. In conclusion, a PTH fusion molecule with GHBP showed bioactivity and delayed clearance. The reduced bioactivity of Fusion-2 compared to PTH and Fusion-1 is compatible with our hypothesis that there is intra-molecular binding to the extracellular domain of the PTH receptor which may result in a store of bioactivity and may explain the greater maximal fold induction in the bioassay despite a reduced EC50. The published serum half-life of PTH 1-34 in rats is 59 minutes1 and so Fusion-1 had greatly delayed clearance. These results validate the approach of generating a long-acting PTH through protein fusion with GHBP. 1. Kostenuik, PJ et al. Journal of Bone and Mineral Research, Volume 22, Number 10, 2007 Sources of Research Support: MRC P2D

MON-536 Improving the Accuracy and Reliability of Parathyroid Hormone Measurements by Establishing a Mass Spectrometric Reference Measurement Procedure

With a rising incidence in renal failure, chronic kidney disease (CKD) has become one of the leading health conditions in the U.S. The earlier stages of CKD generate few symptoms and only until the kidney is significantly impaired do patients begin experiencing signs of renal failure. Parathyroid hormone (PTH) is a key biomarker in patient diagnosis and treatment of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD), as well as hypo/hyperparathyrioidism. Elevated levels of intact PTH (iPTH) have been associated with CKD Stages 3-5. The most recent update of the Kidney Disease Improving Global Outcomes (KDIGO) guideline for CKD-MBD management emphasized the role of PTH as one of the key biomarkers of this disorder. Intact PTH, secreted normally into the blood at 10-65 pg/mL concentration levels, can undergo oxidative or enzymatic metabolic cleavages within parathyroid glands or peripheral tissues to produce N-terminal, C-terminal, and mid-range fragments. The ratio of these PTH fragments to iPTH has been diagnostic for severe or end-stage renal disease, non-dynamic bone disease, and hyperparathyroid-associated bone loss. Current PTH immunoassays demonstrate significant variability with up to a 4.2 fold difference in measurements depending on the method used. This variability can lead to misclassification of patients. Such significant variability has been attributed to differences in antibody specificity, lack of accurate calibration, matrix effects, or interferences. This creates the need for a new accurate and specific method to measure PTH and its fragments. In addition, the low endogenous concentration of PTH in biological matrices requires both, a highly sensitive and specific methodology. The CDC Clinical Standardization Programs in collaboration with the Partnership for the Accurate Testing of Hormones is addressing this need. This work highlights a novel high resolution mass spectrometry-based reference measurement procedure for intact PTH (1-84) and its fragments. Mass spectrometric immunoassay (MSIA) pipette tips were embedded with antibodies for PTH immunocapture. Analytes were eluted from the MSIA tips and analyzed directly with reversed phase ultra-high liquid chromatography high-resolution mass spectrometry (RP-UHPLC-HRMS). Initial results demonstrate the ability to quantify intact PTH and PTH fragments with high specificity. Calibration curves using stable isotope labeled internal standards were generated for iPTH, N-terminal PTH fragments, C-terminal PTH fragments, and mid-region PTH fragments. Initial assessments demonstrate excellent linearity (R2 = 0.991-0.997), reproducibility, and sufficient sensitivity of the UHPLC-HRMS system with a linear range of 15-3000 pg/mL PTH. This method was applied to samples from healthy patients and patients with different CKD stages. PTH and its fragments were detectable in these samples.

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte-Derived Parathyroid Hormone-Related Protein.

Parathyroid hormone-related protein (PTHrP) and parathyroid hormone (PTH) have N-terminal domains that bind a common receptor, PTHR1. N-terminal PTH (teriparatide) and now a modified N-terminal PTHrP (abaloparatide) are US Food and Drug Administration (FDA)-approved therapies for osteoporosis. In physiology, PTHrP does not normally circulate at significant levels, but acts locally, and osteocytes, cells residing within the bone matrix, express both PTHrP and the PTHR1. Because PTHR1 in osteocytes is required for normal bone resorption, we determined how osteocyte-derived PTHrP influences the skeleton. We observed that adult mice with low PTHrP in osteocytes (targeted with the Dmp1(10kb)-Cre) have low trabecular bone volume and osteoblast numbers, but osteoclast numbers were unaffected. In addition, bone size was normal, but cortical bone strength was impaired. Osteocyte-derived PTHrP therefore stimulates bone formation and bone matrix strength, but is not required for normal osteoclastogenesis. PTHrP knockdown and overexpression studies in cultured osteocytes indicate that osteocyte-secreted PTHrP regulates their expression of genes involved in matrix mineralization. We determined that osteocytes secrete full-length PTHrP with no evidence for secretion of lower molecular weight forms containing the N-terminus. We conclude that osteocyte-derived full-length PTHrP acts through both PTHR1 receptor-mediated and receptor-independent actions in a paracrine/autocrine manner to stimulate bone formation and to modify adult cortical bone strength. © 2017 American Society for Bone and Mineral Research.

Synthesis and structural identification of fluorine-18 labeled parathyroid hormone.

Parathyroid hormone (PTH) is an 84 amino acid peptide hormone that plays a key role in bone and mineral metabolism. The biological actions of PTH are mediated via the N-terminal PTH(1-34) fragment, serving as the PTH receptor-binding sequence, and which is therefore used clinically to treat conditions of low bone mass such as osteoporosis. In this study, PTH(1-34) was conjugated with non-radioactive (stable F isotope) N-succinimidyl 4-fluorobenzoate (SFB) leading to three isomeric mono-fluorobenzoated (FBz) PTH followed by Liquid chromatography-Tandem mass spectrometry (LC-MS/MS) assisted structural identification. Corresponding [(18)F]SFB-labeled PTH derivatives were prepared respectively and the Lys(13) site-specific labeled [(18)F]FBz PTH was isolated by HPLC with radiochemical purity >99% and specific activity of 2.78 GBq/µmol, suitable for future application with in vivo pharmacokinetic/pharmacodynamic studies of PTH, using preclinical Positron Emission Tomography Computed Tomography (PET/CT) imaging.

Hypoparathyroidism: Replacement Therapy with Parathyroid Hormone

Hypoparathyroidism (HypoPT) is characterized by low serum calcium levels caused by an insufficient secretion of parathyroid hormone (PTH). Despite normalization of serum calcium levels by treatment with activated vitamin D analogues and calcium supplementation, patients are suffering from impaired quality of life (QoL) and are at increased risk of a number of comorbidities. Thus, despite normalization of calcium levels in response to conventional therapy, this should only be considered as an apparent normalization, as patients are suffering from a number of complications and calcium-phosphate homeostasis is not normalized in a physiological manner. In a number of recent studies, replacement therapy with recombinant human PTH (rhPTH(1-84)) as well as therapy with the N-terminal PTH fragment (rhPTH(1-34)) have been investigated. Both drugs have been shown to normalize serum calcium while reducing needs for activated vitamin D and calcium supplements. However, once a day injections cause large fluctuations in serum calcium. Twice a day injections diminish fluctuations, but don't restore the normal physiology of calcium homeostasis. Recent studies using pump-delivery have shown promising results on maintaining normocalcemia with minimal fluctuations in calcium levels. Further studies are needed to determine whether this may improve QoL and lower risk of complications. Such data are needed before replacement with the missing hormone can be recommended as standard therapy.

Parathyroid hormone and vitamin D-markers for cardiovascular and all cause mortality in heart failure

To investigate levels of vitamin D and parathyroid hormone (PTH) in a population of heart failure (HF) patients, and to evaluate whether vitamin D and PTH are related to prognosis. This was a prospective study of 148 HF outpatients (mean age 68 years, 102 men) with follow-up for mortality after 3½ years. Levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), PTH, 25-hydroxyvitamin D (25-OHD), and several other biomarkers were examined. Mortality and cardiovascular mortality were analysed in multivariable regression analyses adjusting for other independent prognostic variables. Vitamin D deficiency (≤50 nmol/L) was prevalent in 43% of the population; 26% had elevated PTH levels; none had primary hyperparathyroidism. We found a strong and independent significant association of both PTH and vitamin D to mortality, which was independent of other clinically important parameters [NT-proBNP, estimated glomerular filtration rate (eGFR), age, and left ventricular ejection fraction (LVEF)]. Both PTH and vitamin D were also significantly associated with all cause mortality. In an adjusted model, we found a hazard ratio of 1.9 (confidence interval 1.1-3.4) for vitamin D deficiency and 2.0 (1.0-3.8) for the upper median of PTH, respectively. In this relatively small prospective study, PTH and vitamin D were independently associated with all cause and cardiovascular mortality in patients with HF. This was independent of other known risk factors such as eGFR, LVEF, NT-proBNP, and age.

Parathyroid hormone and vitamin D — Markers for cardiovascular and all cause mortality in heart failure

Aims To investigate levels of vitamin D and parathyroid hormone (PTH) in a population of heart failure (HF) patients, and to evaluate whether vitamin D and PTH are related to prognosis. Methods and results This was a prospective study of 148 HF outpatients (mean age 68 years, 102 men) with follow-up for mortality after 3½ years. Levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), PTH, 25-hydroxyvitamin D (25-OHD), and several other biomarkers were examined. Mortality and cardiovascular mortality were analysed in multivariable regression analyses adjusting for other independent prognostic variables. Vitamin D deficiency (≤50 nmol/L) was prevalent in 43% of the population; 26% had elevated PTH levels; none had primary hyperparathyroidism. We found a strong and independent significant association of both PTH and vitamin D to mortality, which was independent of other clinically important parameters [NT-proBNP, estimated glomerular filtration rate (eGFR), age, and left ventricular ejection fraction (LVEF)]. Both PTH and vitamin D were also significantly associated with all cause mortality. In an adjusted model, we found a hazard ratio of 1.9 (confidence interval 1.1–3.4) for vitamin D deficiency and 2.0 (1.0–3.8) for the upper median of PTH, respectively. Conclusion In this relatively small prospective study, PTH and vitamin D were independently associated with all cause and cardiovascular mortality in patients with HF. This was independent of other known risk factors such as eGFR, LVEF, NT-proBNP, and age.

Selected Reaction Monitoring–Mass Spectrometric Immunoassay Responsive to Parathyroid Hormone and Related Variants

Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R(2) = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

An N-Terminal Molecular Form of Parathyroid Hormone (PTH) Distinct from hPTH(1–84) Is Overproduced in Parathyroid Carcinoma

A new parathyroid hormone (PTH) species, the N-terminal PTH form (N-PTH), is distinct from intact human PTH of 84 amino acid residues [hPTH(1-84)] and is recognized in a 3rd-generation assay of "whole" PTH (wPTH; the 1-2 epitope) but not in a 2nd-generation assay of "total" PTH (tPTH; the 12-18 epitope). N-PTH usually represents <15% of wPTH but can be overproduced in severe primary hyperparathyroidism (PHPT) and secondary hyperparathyroidism. We investigated whether N-PTH is also overproduced in parathyroid cancer and whether N-PTH concentration is influenced by calcimimetic therapy. We studied 8 patients with parathyroid carcinoma before and at week 16 of cinacalcet therapy, 6 patients with PHPT, and 6 control individuals. We fractionated sera with HPLC and analyzed fractions with the 2 assays to quantify hPTH(1-84), N-PTH, and non-(1-84) PTH fragments. Half of parathyroid carcinoma patients had an increased wPTH:tPTH ratio [mean (SD), 1.35 (0.29)]; the others had a typical ratio [0.72 (0.12)]. HPLC fractionation of sera from 2 high-ratio patients confirmed N-PTH overproduction [65% (12%) of wPTH]. The N-PTH fraction was <15% of wPTH in PHPT and healthy individuals. Calcimimetic therapy appreciably reduced calcium concentrations in parathyroid carcinoma patients but had little influence on PTH concentration, the wPTH:tPTH ratio, or the PTH HPLC profile. N-PTH is overproduced in some parathyroid cancer patients, but calcimimetic therapy does not influence its production. The clinical implications of this finding in parathyroid carcinoma await additional studies with an emphasis on N-PTH's biological activity and with larger numbers of patients.

Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.

Parathyroid Hormone 7-84 Induces Hypocalcemia and Inhibits the Parathyroid Hormone 1-84 Secretory Response to Hypocalcemia in Rats with Intact Parathyroid Glands

Biologic effects of large C-terminal parathyroid hormone (PTH) fragments, opposite to those of N-terminal PTH, have been demonstrated. C-terminal PTH fragments are co-secreted with N-terminal PTH from the parathyroids. The aim of our study was to examine whether C-terminal PTH 7-84 regulates secretion of PTH 1-84 and affects the expression of genes of relevance for parathyroid function, PTH, calcium-sensing receptor (CaR), PTH type 1 receptor (PTHR1), and PTH-related peptide (PTHrP) genes in rat parathyroid glands. PTH 7-84 induced a significant decrease in plasma Ca2+ in rats with intact parathyroid glands. Despite the reduction of plasma Ca2+, no stimulation of PTH 1-84 secretion took place. Furthermore, the PTH 1-84 secretory response to EGTA-induced acute and severe hypocalcemia was significantly inhibited by PTH 7-84. During recovery from hypocalcemia, plasma Ca2+ levels were significantly lower in the PTH 7-84-treated group, as compared with the vehicle group, and at the same time plasma PTH 1-84 levels were significantly suppressed. The expression of PTH, CaR, PTHR1, and PTHrP genes in the rat parathyroid glands was not affected by PTH 7-84. The peripheral metabolism of PTH 1-84 was not affected by PTH 7-84. PTH 7-84 did not cross-react with the rat bioactive PTH 1-84 assay. In normal rats with intact parathyroid glands, PTH 7-84 inhibited the PTH 1-84 secretory response to hypocalcemia and induced a significant decrease in plasma Ca2+. These effects of PTH 7-84 on PTH 1-84 secretion and on plasma Ca2+ levels were not associated with significant changes in PTH, PTHR1, CaR, and PTHrP gene expressions in the rat parathyroid glands. It is hypothesized that PTH 7-84 regulates PTH secretion via an autocrine/paracrine regulatory mechanism.

Novel Parathyroid Hormone (PTH) Antagonists That Bind to the Juxtamembrane Portion of the PTH/PTH-related Protein Receptor

Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1-34) or PTHrP(1-34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln(10),Ala(12),Har(11),Trp(14),Arg(19) (M)]PTH(1-21), [M]PTH(1-15), and [M]PTH(1-14), and introduced substitutions at positions 1-3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)(1)-para-benzoyl-l-phenylalanine (Bpa)(2)-Deg(3). In HKRK-B7 cells, which express the cloned human PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21), [Deg(1,3),Bpa(2),M]PTH(1-15), and [Deg(1,3),Bpa(2),M]PTH(1-14) fully inhibited (IC(50)s = 100-700 nm) the binding of (125)I-[alpha-aminoisobutyric acid(1,3),M]PTH(1-15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) antagonized the cAMP-agonist action of PTH(1-34), as did PTHrP(5-36) (IC(50)s = 0.7 microm, 2.6 microm, and 36 nm, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg(1,3),Bpa(2), M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) inhibited the agonist actions of [alpha-aminoisobutyric acid(1,3)]PTH(1-34) and [M]PTH(1-14) (IC(50)s approximately 1 microm), whereas PTHrP(5-36) failed to inhibit. [Deg(1,3),Bpa(2),M]PTH(1-14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1-9), in which the PTH(1-9) sequence is covalently linked to the PTHR J domain, as well as that of PTHR(cam)H223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.

Amino-Terminal Parathyroid Hormone Fragment Analogs Containing α,α-di-alkyl Amino Acids at Positions 1 and 3

To define and minimize the N-terminal PTH pharmacophore, the effects of introducing different conformationally constraining di-alkyl amino acids at positions 1 and 3 of PTH(1-14) analogs were studied. Improvements in PTH receptor-binding affinity and signaling potency were found, although some substitutions resulted in partial agonism. The N-terminal portion of parathyroid hormone (PTH) plays a critical role in PTH-1 receptor (P1R) activation. To investigate the mechanisms underlying this action and to minimize the N-terminal PTH pharmacophore, we employed the PTH(1-14) fragment as a scaffold for structure-activity relationship studies, and thus previously found that substitutions of the conformationally constraining, di-alkyl amino acid, alpha-amino-isobutyric acid (Aib), at positions 1 and 3 increase the P1R-binding affinity and signaling potency of the analog approximately 100-fold. Here we extend these findings by investigating the effects of other constrained di-alkyl amino acids at positions 1 and/or 3 on PTH analog activity. The di-alkyl amino acids, 1-aminocycloalkane-carboxylic acid (Ac(x)c, x = 3, 5, or 6) or diethylglycine (Deg), representing alkyl configurations of varying volumes and shape (cyclic and linear), were introduced into the parent peptide, [M]PTH(1-14) (M = Ala(1,3,12),Gln(10),Har(11),Trp(14)), and the analogs were tested for activity in P1R-expressing cells. Relative to the binding affinity and cAMP-stimulating potency of the parent peptide (IC(50) = 27 mM; EC(50) = 220 nM), PTH(1-14) analogs substituted at position 1 exhibited 2- (Ac(3)c) to 60-fold (Ac(5)c) increases in affinity and potency, as measured in LLC-PK1 cells stably expressing the cloned P1R. Combining the substitutions of Ac(5)c(1) and Aib(3) yielded the highest affinity and most potent PTH(1-14) and shorter-length analogs to date: [Ac(5)c(1), Aib(3),M]PTH(1-X) (X = 14, 11, and 10; IC(50)s = 80 nM, 260 nM, and 850 microM; EC(50)s = 1.7 nM, 3.1 nM, and 1.9 microM, respectively). The effects of Ac(6)c(1) were similar to those of Ac(5)c(1). A dissociation of binding affinity and signaling activity occurred with Deg, as [Deg1,3,M]PTH(1-14) was a partial agonist. Constraining the N-terminal PTH backbone conformation with di-alkyl amino acids at positions 1 and 3 may be a general strategy for optimizing and minimizing the PTH pharmacophore; however, inhibitory side-chain effects may be encountered. The new analogs presented should be useful as minimum-length functional probes of the PTH-PTH receptor interaction mechanism.

Relaxant effects of parathyroid hormone and parathyroid hormone-related peptides on oviduct motility in birds and mammals: possible role of nitric oxide

Parathyroid hormone (PTH) and PTH-related peptides (PTHrP) have previously been shown to modulate the contractile state of numerous types of smooth muscle. The effects of N-terminal PTH and PTHrP on spontaneous in vitro contractility of oviducal smooth muscle using tissues from egg-laying Japanese quail (10–15 h post ovulation), 4 and 9 days pregnant mouse uterus were investigated. Myometrial tissues from both species contracted vigorously for several hours, when incubated in organ baths in De Jalon’s solution gassed with 5%CO 2/95%O 2. Contractions were enhanced in high (1.2–2.5 mM) compared with low (0.1–0.5 mM) calcium (Ca) containing media. Bovine PTH(1–34) (bPTH(1–34)), human PTH(1–34 amide) (hPTHrP(1–34) amide), and hPTHrP(1–40) caused similar concentration-related inhibition of contractions in media containing 1.2 mM Ca over a range of 10 −9 to 10 −7 M, whereas C-terminal hPTHrP(107–139) was devoid of such activity. Responses to bPTH(1–34) in 4 and 9-day pregnant mouse tissues were similar but hPTHrP(1–40) showed substantial loss of activity in 9-day, compared with 4-day pregnant mouse tissues. Repeated exposure of mouse uterine tissue to the peptides resulted in desensitisation of responses. The EC 50 responses of mouse tissues were inhibited by the PTH/PTHrP receptor antagonist, hPTHrP(7–34) amide. Responses to bPTH(1–34) were also inhibited by both non-selective and selective neuronal nitric oxide synthase (NOS) inhibitors N ω-nitro- l-arginine methyl ester (0.01–1 mM) and 7-nitroindazole (0.01–10 μM), respectively. Both NOS inhibitors were more effective in inhibiting bPTH(1–34)-induced relaxation in the absence of l-arginine compared with in the presence of 1 mM l-arginine (a NOS substrate) in the incubation media. It is concluded that relaxant responses to N-terminal PTH and PTHrP peptides are well conserved in oviducal and uterine tissues from avian and mammalian species. The results also suggest that NO may be responsible for mediating relaxant activities of these peptides in pregnant mouse uterine tissue.

Autoregulation in the parathyroid glands by PTH/PTHrP receptor ligands in normal and uremic rats

The secretion of parathyroid hormone (PTH) from the parathyroid glands might be regulated by autocrine/paracrine factors. We have previously shown that N-terminal parathyroid hormone-related protein (PTHrP) enhanced the secretory PTH response to low calcium in vivo and in vitro in rat parathyroid glands. N-terminal PTHrP fragments are equipotent to N-terminal PTH as ligands for the PTH/PTHrP receptor that is demonstrated in parathyroid tissue. This supports the possibility that the parathyroid cells respond to PTH/PTHrP receptor ligands and as such are target for an autoregulatory action of PTH and PTHrP. Our aim was to search for the PTH/PTHrP receptor in rat parathyroid glands and to examine the effects of PTHrP 1-40 on PTH secretion in in vivo models of secondary hyperparathyroidism (HPT) in uremic rats. PTH secretion was examined during ethyleneglycol tetraacetic acid (EGTA)-induced hypocalcemia both with and without PTHrP. Five groups, each of six normal rats, received a bolus of increasing doses of 0.1, 1.0, 10, and 100 microg of PTHrP 1-40, or vehicle only. Chronic renal failure (CRF) was induced by 5/6 nephrectomy. One group of 12 CRF rats received a standard diet, while another CRF group of 18 rats received a high phosphorus diet to induce more severe HPT. After 8 weeks of uremia, the rats were infused with EGTA and PTHrP 1-40 or vehicle. The presence of the PTH/PTHrP receptor in the rat parathyroid glands was examined by reverse transcription-polymerase chain reaction (RT-PCR) technique. PTH was measured by a rat PTH assay that does not cross-react with PTHrP. In a dose-related manner, PTHrP enhanced the PTH response to hypocalcemia in normal rats. A similar rate of decrease of plasma Ca++ was induced by EGTA in all experimental groups. In CRF rats, plasma creatinine (0.99 +/- 0.10 mmol/L vs. 0.33 +/- 0.01 mmol/L, P < 0.001) and plasma PTH (226 +/- 32 pg/mL vs. 69 +/- 16 pg/mL, P < 0.001) levels were significantly increased. The CRF rats on high phosphorus diet had significant hypocalcemia (Ca++, 1.04 +/- 0.02 mmol/L vs. 1.28 +/- 0.03 mmol/L, P < 0.001), hyperphosphatemia (3.48 +/- 0.3 mmol/L vs. 2.25 +/- 0.1 mmol/L, P < 0.001) and severe secondary HPT, PTH (984 +/- 52 pg/mL vs. 226 +/- 32 pg/mL, P < 0.001) compared to CRF rats on a standard phosphorus diet. The maximal PTH response to hypocalcemia was enhanced in CRF rats (maximum PTH 382 +/- 58 pg/mL vs. 196 +/- 29 pg/mL in normal rats, P < 0.01) and further enhanced by PTHrP 1-40 to 826 +/- 184 pg/mL (P < 0.01). The secretory capacity of the parathyroid glands in response to low Ca++ was severely diminished in uremia. In CRF rats given a high phosphorus diet, the basal PTH levels were at the upper part of the calcium/PTH curve, and the induction of more marked hypocalcemia did not stimulate PTH secretion further (maximum PTH 1475 +/- 208 pg/mL vs. basal 1097 +/- 69 pg/mL, NS). PTHrP, however, further enhanced the maximal PTH levels significantly (maximum PTH 3142 +/- 296 pg/mL, P < 0.01). The presence of the PTH/PTHrP receptor in the rat parathyroid glands was confirmed by RT-PCR technique. PTHrP enhanced significantly, in a dose-related manner, the low Ca++-stimulated PTH secretion in normal rats. The PTH/PTHrP receptor is present in rat parathyroid glands. The impaired secretory capacity of the parathyroid glands in uremic rats is significantly enhanced by PTHrP. An autocrine/paracrine role in the parathyroid glands of the PTH/PTHrP receptor targeting peptides, PTHrP and PTH, is suggested. Thus, it is hypothesized that PTH during hypocalcemia might have a positive auto-feedback regulatory role on its own secretion.

Characterization of the human and mouse genes encoding the tuberoinfundibular peptide of 39 residues, a ligand of the parathyroid hormone receptor family.

The polypeptide TIP39 (tuberoinfundibular peptide of 39 residues) is a potent activator of the parathyroid hormone (PTH)-2 receptor (P2R) and an antagonist of the PTH-1 receptor (P1R). To clarify its possible physiological function(s), we studied its interaction with the human P1R and P2R and examined the expression of TIP39 in man and mouse. To find out possible sites of this ligand interaction in the organism, we identified the genes encoding the TIP39 protein precursors of Homo sapiens and Mus musculus in the databases of the human and mouse genome projects respectively. We then obtained the full-length cDNAs of both species by RACE-PCR. The deduced TIP39 preprohormones consist of an N-terminal 30 amino acid (aa) signal peptide followed by a 29 aa TIP39 precursor-related peptide, an Arg-Arg processing site, and the actual 39 aa TIP39 sequence. The first 23 aa of the actual TIP39 sequence, thought to contain the P2R receptor activation site, are identical in man and mouse and thus phylogenetically conserved. By contrast, the 16 aa C-terminal portion showed a higher degree of diversity (75% aa identity). By using RT-PCR, TIP39 was found to be highly expressed in human central nervous system tissues, trachea, fetal liver, and, to a lesser degree, in human heart and kidney. Using in situ hybridization, TIP39 mRNA expression was revealed in various areas of the mouse brain. In a homologous human cell model using human embryonic kidney 293 cells stably transfected with human P1R and P2R, human TIP39 did bind to P1R with moderate affinity (IC(50) approximately 10(-7)-10(-6 )M), but showed higher affinity binding to P2R (IC(50) approximately 10(-8)M), comparable to the affinity of human N-terminal PTH (hPTH(1-34)) to this receptor. In P2R-transfected cells, the cAMP pathway was activated more efficiently ( approximately 10-fold) by TIP39 as a ligand compared to hPTH(1-34). In P1R-transfected cells, only hPTH(1-34) but not TIP39 was able to elicit a cAMP response, but TIP39 was able to directly antagonize the cAMP-stimulating effect of hPTH(1-34) on this receptor. In conclusion, we could show a possible function of TIP39 for the human organism as a potent activator of P2R (e.g. in brain) as well as an antagonist of the action of PTH and/or PTH-related protein on P1R (e.g. in bone and kidney). The physiological role of TIP39 in calcium metabolism with regard to these actions remains to be determined. The tools developed in this work will allow us to investigate the possible role of TIP39 as a locally or systemically secreted ligand modulating the function of the PTH receptor family.

Developments in parathyroid hormone and related peptides as bone-formation agents.

Osteoporosis is a major and growing healthcare concern. When administered by daily injection, parathyroid hormone (PTH) and its N-terminal fragments and analogs are potent bone-formation agents. Teriparatide, recombinant human PTH(1-34), is likely to be the first anabolic agent approved for treating osteoporosis, despite inducing osteosarcomas in rats. Native PTH and other PTH fragments and analogs are also in development. N-terminal fragments sometimes differ in activity from the native hormone, however, and the C-terminal region of PTH, acting through a receptor different from the classical PTH-1 receptor, initiates a variety of distinct biological activities. In particular, the C-terminal region of PTH, by promoting bone-cell apoptosis, may be important in opposing the anti-apoptotic effects of teriparatide in these cells, thereby maintaining normal bone-cell turnover. Because of these differences, care must be taken to consider the effects of native PTH and N-terminal PTH fragments and analogs separately.

Parathyroid Hormone (PTH)-(1–14) and -(1–11) Analogs Conformationally Constrained by α-Aminoisobutyric Acid Mediate Full Agonist Responses via the Juxtamembrane Region of the PTH-1 Receptor

The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated whether substitution of the sterically hindered and helix-promoting amino acid alpha-aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH(2) ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib(1,3),M]PTH(1-14) was approximately 100-fold more potent than [M]PTH(1-14) (EC(50) = 1.1 +/- 0.1 and 100 +/- 20 nm, respectively), approximately 100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib(1,3),M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11) (EC(50) 4 +/- 1 nm versus 3 +/- 1 microm). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib(1,3),M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) (EC(50) = 0.7 +/- 0.2 versus 40 +/- 2 nm) and 1,000-fold more potent than PTH(1-34) (EC(50) = 700 nm). [Aib(1,3),M]PTH(1-14), but not PTH(1-34), inhibited the binding of (125)I-[Aib(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH(1-21)NH(2) to hP1R-delNt (IC(50) = 1,600 +/- 200 nm). The Aib(1,3) substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). The overall data thus suggest that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an alpha-helix, upon interaction with the activation domain of the PTH-1 receptor.

Influence of a low calcium and phosphorus diet on the anabolic effect of human parathyroid hormone (1-38) in female rats

Parathyroid hormone (PTH) or synthetic N-terminal PTH fragments administered intermittently have been established as anabolic agents in animal and human bones. In the present study, the influence of a low calcium diet on the anabolic effect of human PTH(1-38) [hPTH(1-38)] was investigated. Forty-eight 10-week-old female Sprague-Dawley rats were randomly assigned to a diet with a low calcium content (LCa) or a diet with the recommended amount of calcium (RCa). After an adaptation period of 15 days, the rats were randomly assigned to hPTH(1-38) treatment (+LCa/+RCa) or vehicle only (−LCa/−RCa) for an additional 14 days. Total bone mineral density (BMD) values of several bones were determined using quantitative computed tomography and from ratios of ash weight to volume. Biomechanical competence of the fourth lumbar vertebrae and of the right femora was assessed. An anabolic effect could be detected in both PTH-treated groups. However, the bones of the +LCa group showed significantly lower BMD and also a diminished increase in maximal breaking force compared with those of the +RCa group. The study demonstrates that the anabolic effect of hPTH(1-38) is blunted by the LCa diet. This suggests that, during PTH treatment, dietary calcium intake is critical.

C-terminal parathyroid hormone-related protein increases vascular endothelial growth factor in human osteoblastic cells.

The N-terminal region of parathyroid hormone (PTH) and PTH-related protein (PTHrP) interacts with a common PTH/PTHrP receptor in osteoblasts. These cells synthesize PTHrP, but its role in bone turnover is unclear. Intermittent treatment with N-terminal PTHrP or PTH stimulates bone growth in vivo, possibly by increasing local bone factors. In addition, C-terminal PTHrP (107-139), which does not bind to the PTH/PTHrP receptor, appears to affect bone resorption in vivo and in vitro, although its effect on bone formation in vivo remains controversial. Bone angiogenesis is an often overlooked but critical event in the process of bone remodeling. Recently, PTH (1-34) has been shown to induce gene expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, by osteoblastic cells. However, no data are available on the effect of PTHrP (107-139) on VEGF expression in these cells. Using semiquantitative reverse transcription followed by PCR, we found that PTHrP (107-139), between 10 nM and 1 pM, increased VEGF mRNA in human osteoblastic (hOB) cells from trabecular bone. This effect of this agonist, at 10 nM, was maximal (fivefold for VEGF(165), and twofold for VEGF(121), compared to control) within 1 to 4 h. This effect was similar to that induced by PTHrP (1-34) in these cells, as well as in human osteosarcoma MG-63 cells, using Northern blot analysis. Moreover, the effect of both peptides, added together at 100 pM, was not higher than that observed with each peptide alone in hOB cells. The effects of PTHrP (107-139) and that of PTHrP (1-34) were abolished by actinomycin D in hOB cells. In these cells, the protein kinase C inhibitor staurosporine, but not the protein kinase A inhibitor H89, inhibited the increase in VEGF mRNA induced by 10 nM PTHrP (107-139). PTHrP (107-139), at 10 nM, also stimulated cytosolic VEGF immunostaining in hOB cells, and VEGF secretion into the medium conditioned by hOB or MG-63 cells for 24 h, which was (ng/mg protein): 10 +/- 1 or 5 +/- 3 (control), respectively, and 21 +/- 1 or 11 +/- 2 (PTHrP [107-139]-stimulated), respectively. Furthermore, medium conditioned by these cells for 24 h in the presence of 10 nM PTHrP (107-139), with or without 10 nM PTHrP (1-34), increased about 30% bovine aortic endothelial cell (BAEC) growth at 48 h. This effect was inhibited by adding a specific anti-VEGF antibody to the BAEC incubation medium. These findings demonstrate that the C-terminal domain of PTHrP induces expression and secretion of VEGF, a main angiogenic factor, in hOB cells and MG-63 cells. This relationship between PTHrP and VEGF has potential implications for both bone vascularization and bone formation, and neoangiogenesis in PTHrP-producing tumors.